Molecular techniques in haematopathology: what and how?

Abstract

Here we review the 'what and how' of molecular techniques used in the context of haematopathological diagnostics of both lymphoid and myeloid neoplasms. Keeping in mind that the required resources for molecular testing are not universally available, we will not only discuss novel and emerging techniques that allow more high-throughput and sophisticated analyses of lymphoid and myeloid neoplasms, but also the more classical, low-cost alternatives and even some workarounds for molecular testing approaches. In this review we also address other key aspects around molecular techniques for haematopatholgy diagnostics, including preanalytics, data interpretation, and data management, bioinformatics, and interlaboratory precision and performance evaluation.

Figure 1

Schematic representation of various molecular alterations relevant to lymphoid and myeloid neoplasms and detection techniques. Sensitivity levels vary depending on the techniques used. For example, Sanger sequencing is unable to detect subclonal (<20%) single‐nucleotide changes, sequencing‐based approaches, and karyotyping may miss subclonal copy number alterations. A few selected examples of single‐gene tests are shown in the figure. Created with BioRender.com. ASO PCR, allele‐specific oligonucleotide PCR; MLPA, multiplex ligation‐dependent probe amplification; OGM, optical genome mapping; qPCR, quantitative/real‐time PCR; WGS, whole genome sequencing.

Figure 2

IG/TR rearrangement analysis and applications. (A) B and T cells carry highly specific antigen receptors, termed immunoglobulin (IG) and T‐cell receptors (TR), respectively. These antigen receptors, here exemplified for the immunoglobulin, consist of unique variable domains (purple colour variants) created by genetic rearrangements of V, D, and J genes on a per‐cell basis. The resulting V(D)J rearrangement is referred to as a clonotype that functions as a molecular fingerprint, especially through its CDR3 region. (B) IG/TR gene analysis of lymphoid cells discloses the heterogeneity of lymphoid cells, with a spectrum ranging from completely diverse (polyclonality) to fully identical (clonality). Clonality assessment can be done by fragment/Gene Scan (GS) analysis or through NGS methods. (C) IG/TR gene analysis can also be applied for prognosis in CLL through identification of somatically mutated vs unmutated cases (upper graphic) or even stereotypic subsets with a characteristic CDR3 (middle graphic). Another application concerns the analysis of IG/TR clonotypes for MRD purposes (lower graphic).

Figure 3

Schematic work‐flow of sequencing platforms. (A) Sanger sequencing is based on the selective incorporation of chain‐terminating dideoxynucleotides (ddNTPs) by DNA polymerase during DNA replication. During the chain termination PCR, DNA polymerase incorporates deoxynucleotides (dNTPs) into the growing DNA strand. When a fluorescently labelled ddNTP is incorporated, the strand elongation is terminated as ddNTPs lack a 3′‐OH group required to form a phosphodiester bond with the next nucleotide. The resulting terminated DNA fragments of various lengths are then separated by capillary electrophoresis. The fluorescence of the terminating ddNTPs is detected, and the DNA sequence is determined by the order of the fluorescence peaks recorded, which corresponds to the nucleotide sequence in the DNA strand. (B) Next‐generation sequencing (NGS) is a high‐throughput methodology that allows for the simultaneous sequencing of millions of DNA targets from multiple patient samples. Here, Illumina sequencing chemistry is depicted. During NGS library preparation, DNA is fragmented into smaller pieces. Sequencing adapters and indexes are ligated to the ends of these DNA fragments. The indexes enable sample identification within pooled data. Depending on whether targeted NGS or whole‐genome sequencing (WGS) is desired, a target enrichment step may be included. Libraries from multiple samples are pooled together and loaded onto a flow cell coated with oligonucleotides complementary to the sequencing adapters. The library DNA is then immobilized on the surface and undergoes bridge amplification, generating clusters of amplified DNA. These clusters augment the fluorescent signal during sequencing cycles. During sequencing, fluorescently labelled nucleotides are added. Each incorporated nucleotide emits a fluorescent signal that is detected and recorded. This process is repeated for each nucleotide in the sequence. This sequencing is performed in parallel across millions of clusters, enabling the massive parallel sequencing of multiple targets from multiple patients. (C) Error‐corrected NGS enhances sequencing accuracy by incorporating molecular barcodes and consensus sequence analysis to correct errors introduced during sequencing and amplification, making it especially useful in MRD assessment. During library preparation, molecular barcodes of unique molecular identifiers (UMIs) are attached to each DNA fragment before amplification. After sequencing, reads sharing the same UMIs are grouped and analysed. This allows for the identification of true mutations (e.g., “A” in all reads of the orange read group) and distinguishes them from random errors (e.g., “G”, “T”, “A” in a subset of reads in the green and red read groups). (D) Long‐read sequencing. Various platforms exist and Oxford Nanopore technology is depicted here. Nanopore array is submerged in an electrolyte solution and an electric potential is applied across the membrane. Double‐stranded DNA is unwound by the motor protein and the single‐strand molecule passes through the nanopore one at a time. As each nucleotide passes through the nanopore, it causes characteristic disruptions in the ionic current flowing through the pore. These disruptions generate a unique signal for each nucleotide. The device detects and records these electrical signals in real‐time, producing raw signal data that corresponds to the nucleotide sequence of the DNA molecule. A, B, and D adapted from “Sanger sequencing”, “Next generation sequencing (Illumina)”, and “Nanopore sequencing”, by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender‐templates.